CRISPR RNAs Trigger Innate Immune Responses in Human Cells
Open accessAbstract
Here, we report that CRISPR guide RNAs (gRNAs) with a 5′-triphosphate group (5′-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5′-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5′-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4 + T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5′-hydroxyl group are much more efficient than in vitro–transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.
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Chemically synthesized gRNAs with a 5'-hydroxyl group are more efficient and less immunogenic than in vitro-transcribed sgRNAs in human and other mammalian cells.
“The authors conclude that chemically synthesized gRNAs with a 5 -hydroxyl group are more efficient and less immunogenic than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells.”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [38]
In primary human CD4+ T cells, delivery of 5'-ppp gRNAs resulted in higher expression of innate response-related genes and IFN-β secretion compared to 5'-hydroxyl gRNAs.
“The results showed that there was a higher expression of innate responserelated genes and IFN-β secretion in groups that received 5 -ppp gRNAs.”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [38]
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- Authors
- Kim, S., Koo, T., Jee, H.-G., Cho, H.-Y., Lee, G., Lim, D.-G., Shin, H. S., & Kim, J.-S.
- Affiliations
- Institute for Basic Science (KR)
- National Medical Center (KR)
- Seoul National University (KR)
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2018-02-22
- Volume
- 28
- Issue
- 3
- Pages
- 367-373
- ISSN
- 1088-9051 (ISSN-L)
- 1549-5469
- PMC
- PMC5848615
- OpenAlex
- W2792675259
- Cited by
- 222
- References
- 29
- Citations by year
- 2026: 14 · 2025: 19 · 2024: 22 · 2023: 22 · 2022: 25 · 2021: 35 · 2020: 43 · 2019: 28 · 2018: 13
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Funders
- Institute for Basic Science (IBS-R021-D1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR guide RNA, 5'-triphosphate RNA, RIG-I, type I interferon response, cell death, phosphatase treatment, Cas9, CRISPR/Cas12a, targeted mutagenesis, CD4+ T cells, in vitro transcription, sgRNA design
- MeSH terms
- DEAD Box Protein 58, Gene Editing, RNA, Guide, CRISPR-Cas Systems, Animals, Humans, Immunity, Innate, Interferon Type I, CD4-Positive T-Lymphocytes, Mice, HEK293 Cells, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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